Rhodococcus erythropolis capable of converting quininone into (S)-3-quinuclidinol and conversion method

A technology of Rhodococcus erythropolis and quinine alcohol, which is applied in the field of bioengineering to achieve the effects of easy cultivation, high yield and stable catalysis

Active Publication Date: 2014-04-02
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

At present, there is no report on the synthesis of (S)-3-quinine alcohol by biological methods in China

Method used

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  • Rhodococcus erythropolis capable of converting quininone into (S)-3-quinuclidinol and conversion method
  • Rhodococcus erythropolis capable of converting quininone into (S)-3-quinuclidinol and conversion method
  • Rhodococcus erythropolis capable of converting quininone into (S)-3-quinuclidinol and conversion method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Screening and identification of strains

[0051] 1.1 Medium

[0052] Inorganic salt medium: K 2 HPO 4 ·3H 2 O 1.0g, Na 2 HPO 4 ·3H 2 O 1.0g, (NH 4 ) 2 HPO 4 2.0g, NaNO 3 2.0g, MgSO 4 ·7H 2 O 0.2g, CaCl 2 2H 2 O 10mg, FeSO 4 ·7H 2 O 1.0mg, ZnSO 4 0.1mg, distilled water 1000mL.

[0053] A certain concentration of quinine hydrochloride was added to the inorganic salt medium to prepare the corresponding enrichment medium.

[0054] 1.2 Isolation and purification of strains

[0055] Put 5 g of the collected sample into a 250 mL shaker flask containing 50 mL of sterile water, add glass beads, oscillate on a shaking table at 250 r / min for 2 hours, and let it stand for 3-5 minutes. After the soil particles settle, take out 2 mL of the supernatant and add Into 50mL liquid enrichment medium (containing quinine hydrochloride 5g / L), 30°C, 200r / min shaker culture for 3-4 days, transfer to the next batch of enrichment medium according to 2% inoculum...

Embodiment 2

[0059] Embodiment 2: Preliminary optimization of fermentation conditions of Rhodococcus erythropolis WY1406

[0060] 1 Single factor experiment of carbon source

[0061] The addition amount of different carbon sources in the fermentation medium was 15g / L.

[0062] Table 1, the reaction of different carbon source fermentation bacteria transforming quinine into (S)-3-quinine alcohol

[0063]

[0064] From image 3 It can be seen from the results that when glucose and sucrose were used as carbon sources, the cell growth was the best. Among them, when sucrose and maltose were used as carbon sources, the conversion effect of the bacteria to quinine was the best. Considering the growth of bacteria and the conversion of substrates, sucrose is the optimal carbon source.

[0065] 2 Single factor experiment of nitrogen source

[0066] The addition amount of different nitrogen sources in the fermentation medium was 10g / L.

[0067] Table 2, the reaction of different nitrogen sour...

Embodiment 3

[0070] Embodiment 3: strain conversion quinine ketone is the conversion method of (S)-3-quinine alcohol

[0071] 1 Preparation of seed solution

[0072] Inoculate Rhodococcus erythropolis WY1406 into the sterilized liquid medium, culture at 30° C. for 24 h, and shaker speed 200 r / min to obtain seed liquid.

[0073] The liquid medium is configured according to the following proportions: glucose 10g / L, peptone 5g / L, yeast extract 5g / L, sodium chloride 1g / L, pH 7.0.

[0074] 2 Fermentation expansion culture

[0075] The seed liquid obtained in step 1 is inoculated into the fermentation medium according to the inoculation amount of 1% to carry out fermentation culture. Culture conditions: 30°C, shaker speed 200r / min, fermentation time 24h, the fermentation broth of Rhodococcus erythropolis is obtained.

[0076] The composition of the fermentation medium is: sucrose 15g / L, yeast extract 7g / L, peptone 3g / L, potassium dihydrogen phosphate 1g / L, dipotassium hydrogen phosphate 1g / L,...

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Abstract

The invention discloses a Rhodococcus erythropolis WY1406 and a production method for (S)-3-quinuclidinol through fermentation of quininone by using Rhodococcus erythropolis WY1406. According to the invention, quininone hydrochloride is used as a sole carbon source for the strain of Rhodococcus erythropolis WY1406 which is screened from soil in a farm orchard in Xiqing District, Tianjin City, China, and is preserved in China General Microbiological Collection Center with an accession number of CGMCC No. 5096. The production method comprises the following steps: with the Rhodococcus erythropolis WY1406 as a strain, carrying out primary seed culture and secondary fermentation amplification culture; taking a fermentation culture solution, collecting thalli through centrifugation, washing the thalli and re-suspending the thalli in a buffer solution; adding the substrate quininone hydrochloride, adding glucose and carrying out centrifugation after a reaction for 10 to 48 h; adjusting the pH value of a supernatant until the supernatant is alkaline; drying the supernatant through pressure reduced spinning; subjecting an obtained residue to solid-liquid extraction with an organic solvent; and drying an obtained filtrate through spinning so as to obtain (S)-(+)-3-quinuclidinol. According to the invention, the product (S)-(+)-3-quinuclidinol has a yield of 95%, a purity of 95% and an e.e value of more than 99%; the production method is simple to operate, has low energy consumption, accords with requirements for green chemistry and is applicable to large-scale bioconversion production for (S)-(+)-3-quinuclidinol.

Description

technical field [0001] The invention relates to the field of bioengineering, and relates to a bacterial strain for fermenting and synthesizing (S)-(+)-3-quinine alcohol and a production method thereof. Background technique [0002] [0003] (S)-(+)-3-Quinuclidinol, English name: (S)-(+)-3-Quinuclidinol, chemical name: (S)-(-)-1-azabicyclo[2.2.2] Octan-3-ol, molecular formula C 7 h 13 NO, the molecular weight is 127.18, the CAS number is 34583-34-1, the pure product of (S)-3-quinine alcohol is white crystal, the melting point is 221-224°C, the boiling point is 120°C, and the specific rotation is +44.5°( c=3g / 100ml, 1N HCl), the solubility in water is 100g / 100mL. (S)-(+)-3-quinine alcohol is a very promising and important chiral building block, which can be introduced into the desired chiral drug intermediate through reactions such as the replacement of hydroxyl groups. It can be used for the synthesis of many active drugs. Such as serotonin receptor antagonist drugs (F...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P17/18C12R1/01
Inventor 王玉李建煚吴洽庆朱敦明
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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